JP7314161B2 - IL-13 receptor α2-targeted T-cell immunotherapy directed by zetakine - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Application No. 62/643,139, entitled "Zetakine-Targeted IL-13 Receptor α2-Targeting T Cell Immunotherapy," filed March 14, 2018, which is expressly incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING This application has been filed with the Sequence Listing in electronic form. This sequence listing is provided as a file of approximately 35 kb created on March 12, 2019 under the file name SCRI179WOSEQLIST. The information contained in this electronic form of the Sequence Listing is expressly incorporated herein by reference in its entirety.

Some of the embodiments of the methods and compositions provided herein provide cell-based methods of targeting cancer cells (such as solid tumor cells) using cells having the receptor zetakine targeted by a cell membrane-bound IL-13 mutein, e.g. Including immunotherapy. In some embodiments, the receptor comprises a spacer region, e.g., a spacer region designed, particularly selected to have a predetermined length, to provide the receptor with certain desirable properties.

Despite a better understanding of brain cancer, mortality rates have remained constant over the past decade, and innovative new treatments are urgently needed. T-cell immunotherapy is now a promising cancer therapy, supported by remarkable clinical data showing complete remissions in patients with B-cell malignancies following administration of CD19-targeted CAR-expressing T cells. However, there remains a need for improved and additional T cell immunotherapies.

Some of the embodiments of the methods and compositions provided herein are nucleic acids encoding zetakine, a receptor targeted by a cell membrane-bound IL-13 mutein, comprising:
the receptor zetakine is
an extracellular domain containing a mutein and spacer of IL-13;
a transmembrane domain; and an intracellular signaling region,
The nucleic acid is characterized in that said spacer is located between said mutein and said transmembrane domain.

In some embodiments, the IL-13 mutein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:16. In some embodiments, the IL-13 mutein comprises the amino acid sequence of SEQ ID NO:16.

In some embodiments, said spacer is a peptide spacer.

In some embodiments, the peptide spacer is 110 amino acids or less in length and is 1 or more amino acids in length or 2 or more amino acids in length, such as 15 amino acids or less in length and 1 or more or 2 amino acids in length or more.

In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (short spacer), an IgG4 hinge-CH3 spacer (medium length spacer), or an IgG4 hinge-CH2-CH3 spacer (long spacer).

In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge-CH3 spacer (medium length spacer).

In some embodiments, the spacer comprises, consists of, or consists essentially of the IgG4 hinge-CH2-CH3 spacer (long spacer).

In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:10.

In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:11.

In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain (CD28tm).

In some embodiments, the intracellular signaling domain comprises a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and combinations thereof, in combination with all or part of the CD3ζ domain. In some embodiments, the intracellular signaling region comprises a signaling functional portion of the CD3ζ domain and a co-stimulatory functional portion of the 4-1BB domain.

Some embodiments further comprise sequences encoding markers. In some embodiments, the marker comprises a truncated form of a cell surface receptor, and the marker may be EGFRt.

Some embodiments further comprise a transgene encoding dihydrofolate reductase configured to be selectable by methotrexate. In some embodiments, the transgene encoding dihydrofolate reductase is a dihydrofolate reductase double mutant (DHFRdm). In some embodiments, the dihydrofolate reductase double mutant comprises an L22F amino acid mutation and an F31S amino acid mutation.

Some embodiments further comprise a sequence encoding a ribosome skipping sequence.

In some embodiments, said ribosome skip sequence comprises P2A or T2A.

Some of the embodiments of the methods and compositions provided herein include expression vectors comprising nucleic acids according to any of the embodiments provided herein. In some embodiments, said vector is a viral vector. In some embodiments, the vector is a lentiviral or adenoviral vector.

Some of the embodiments of methods and compositions provided herein comprise a chimeric receptor polypeptide encoded by a nucleic acid according to any of the embodiments provided herein.

Some of the embodiments of methods and compositions provided herein comprise a host cell comprising nucleic acid according to any of the embodiments provided herein.

In some embodiments, the host cell is a T cell or progenitor T cell.

In some embodiments, said host cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some embodiments, said CD8+ cytotoxic T lymphocytes are central memory T cells, and said central memory T cells are CD45RO+, CD62L+ and CD8+.

In some embodiments, said host cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells and bulk CD4+ T cells. In some embodiments, said CD4+ helper lymphocytes are naive CD4+ T cells, and said naive CD4+ T cells are CD45RA+, CD62L+ and CD4+, and CD45RO-.

In some embodiments, said host cell is a progenitor T cell.

In some embodiments, said host cells are hematopoietic stem cells.

Some of the embodiments of the methods and compositions provided herein include compositions comprising host cells according to any of the embodiments provided herein and a pharmaceutically acceptable excipient.

Some of the embodiments of the methods and compositions provided herein are methods of making a host cell, such as a host cell according to any of the embodiments described herein, comprising:
introducing a nucleic acid according to any of the embodiments described herein into a lymphocyte;
culturing said lymphocytes in the presence of anti-CD3 and/or anti-CD28 antibodies and at least one homeostatic cytokine; and selecting said lymphocytes with a selection reagent configured to selectively enrich for cells transduced with said nucleic acid or a vector comprising said nucleic acid.

In some embodiments, the selection reagent comprises methotrexate.

In some embodiments, the lymphocytes have a CD45RA-, CD45RO+ and CD62L+ phenotype. In some embodiments, said lymphocytes are CD8+ or CD4+.

In some embodiments, said cytokine is IL-15, IL-7 and/or IL-21.

Some embodiments further comprise introducing a second polynucleotide encoding a marker protein into said host cell. In some embodiments, said marker protein is EGFRt.

Some of the embodiments of the methods and compositions provided herein include host cells according to any of the embodiments described herein for use in medicine or in the treatment or suppression of cancers or solid tumors that express IL-13 receptor alpha 2 (IL-13Ra2). In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is an IL13Rα-positive malignancy. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM).

Some of the embodiments of the methods and compositions provided herein include methods of treating, suppressing or ameliorating cancer in a subject comprising administering a host cell according to any of the embodiments provided herein to a subject in need of treatment, suppression or amelioration of cancer. In some embodiments, the cancer is an IL13Rα-positive malignancy. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is glioma or glioblastoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

Some embodiments further comprise administering an adjunctive therapy selected from chemotherapy and radiation therapy. In some embodiments, the chemotherapeutic agent is electrochemotherapy, an alkylating agent, an antimetabolite (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexine). sart, pemetrexed (Alimta®, pentostatin and thioguanine), antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators or checkpoint inhibitors (checkpoint kinases CHK1, CHK2).

Schematic representation of nucleic acids encoding specific IL-13 (E13Y) zetakine CARs with different spacer regions. Nucleic acids encoding the IL-13 (E13Y) zetakine CAR included a leader sequence (EF1p); a polynucleotide encoding a mutein of IL-13 (E13Y); a polynucleotide encoding any one of three spacers; a polynucleotide encoding a CD28tm transmembrane sequence; The three spacers were a short 'S' spacer containing a modified IgG4 hinge; a medium-length 'M' spacer containing a modified IgG4 hinge and an immunoglobulin CH3 region (IgG4 hinge-CH3); and a long 'L' spacer containing a modified IgG4 hinge and an immunoglobulin CH2 domain and an immunoglobulin CH3 domain (IgG4 hinge-CH2-CH3). The L spacer contained two mutations (L235D, N297Q) in the CH2 domain. In addition, CAR contained a self-cleaving ribosomal skipping sequence 2A peptide (T2A). In some constructs, the lentivector further contained a transgene encoding a double mutant of dihydrofolate reductase (DHFRdm) configured to be selectable by methotrexate.

Each step of the method for generating chimeric receptor-expressing T cells is shown, including the CD8+ cell selection step and culture conditions. Selection of CD8 cells and culture conditions: CD8 T cells were selected from PBMC using CD8 microbeads. After stimulation of the selected cells with CD3/CD28 microbeads, lentivirus was used to transduce the construct of FIG. In several replicate experiments (*), the CD8 T cells were transduced with a construct further comprising T2A-DHFRdm inserted in-frame downstream of EGFRt to co-express T2A-DHFRdm, allowing selection of MTX-resistant T cells co-expressing IL13 zetakine CAR in functionally relevant amounts.

Flow cytometry data staining for surrogate marker (EGFRt) expression in CD8+ T cells with CARs containing S, M or L spacers are shown.

The results of analysis of IL13Ra2 expression in U87, U251T and DAOY tumor cell lines as target cells are shown.

Shown are graphs analyzing the specific lysis of target cells (upper panel: U87; middle panel: U251T; and lower panel: DAOY) in the presence of effector CD8+ T cells containing CARs with S, M or L spacers, with the indicated ratios. Data represent the mean±SD of 3 different donors.

Shown are graphs analyzing the production of cytokines by CD8+ T cells containing CARs with S, M or L spacers in the presence of target cells (upper row: U87; middle row: U251T; and lower row: DAOY). Error bars indicate mean±SD.

FIG. 10 shows a flux analysis graph showing the time course of tumor burden in mice injected with T cells containing IL13 zetakine CARs with M or L spacers at various doses.

Kaplan-Meier survival curves for mice injected with various doses of T cells containing IL13 zetakine CARs with M or L spacers.

Steps of a method for generating and analyzing cells containing IL13 zetakine CAR are shown.

Flow cytometry data analyzing the expression of CD4, CD8, EGFTt and zetakine (stained with anti-IL13 antibody) in cells containing IL13 zetakine CAR with M-spacer or mutated L-spacer are shown.

Shown are the results of analysis of the specific lysis of target cells in the presence of effector CD4+/CD8+ T cells containing CARs with M spacers or L spacers at the indicated ratios.

FIG. 4 shows the results of analysis of tumor progression by flux measurements in mice treated with cells containing IL13 zetakine CARs with M or L spacers.

Shown are the results of analysis of the specific lysis of target cells in the presence of effector CD4+/CD8+ T cells containing CARs with M spacers or L spacers at the indicated ratios.

Figure 2 shows analysis results of cytokine release assay in CD4+/CD8+ T cells containing IL13 zetakine CAR with M spacer or L spacer.

Some of the embodiments of the methods and compositions provided herein relate to IL13 zetakine, a chimeric antigen receptor (CAR) capable of specifically binding to interleukin-13 receptor alpha2 subunit (IL-13Rα2) or configured to specifically bind to IL-13Rα2. Some embodiments include a nucleic acid encoding such a CAR, a host cell comprising the CAR, and a therapeutic method utilizing the host cell comprising the CAR, which can treat or inhibit, for example, an interleukin-13 receptor α2 subunit (IL-13Rα2)-mediated disease or condition (e.g., cancer such as IL-13Ra2-positive malignancies, including glioma and glioblastoma) by providing the host cell as a pharmaceutical to a subject in need thereof. .

IL-13Rα2 has been previously reported to be overexpressed in metastatic or late-stage Basal-like breast cancer (BLBC) (Papageorgis et al. Breast Cancer Research, 2015; 17 (1); this document is expressly incorporated herein by reference in its entirety). A correlation has also been found between prediction of progression-free survival and high expression of IL-13Rα2, based on publicly available data. High IL-13Rα2 expression has also been observed in BLBC subtypes that tend to spread rapidly to the lungs. In addition, IL-13Rα2 has been found to stimulate human glioma cell proliferation and also metastasis via the Src/PI3K/Akt/mTOR signaling pathway (Tu et al. Tumor Biol. 2016 Nov; 37(11):14701-14709; which is expressly incorporated herein by reference in its entirety). IL13Ra2-targeted therapies, such as therapies using chimeric receptors, have been previously reported (see, for example, Brown et al Clin Cancer Res 2015; Brown et al N Engl J Med 2016; Brown et al Mol Ther 2017; WO2014072888(A1); 2) Antibodies and antibody-drug conjugates have been reported; these references are expressly incorporated herein by reference in their entirety). Such therapies include therapies using binding domains or antigen recognition domains (eg, zetakine, etc.) based on IL13 variants (eg, E13Y) or therapies involving these domains. Interleukin-13 receptor α-chain variant 2 (IL-13Rα2) is a desirable target for adoptive transfer T-cell therapy. More specifically, IL-13Rα2 expression is used as a prognostic marker for patient survival in glioblastoma multiforme (GBM) and has been implicated in promoting tumor progression.

IL-13Ra2 is expressed in more than 80% of high-grade gliomas, such as glioblastoma multiforme, but in these tumors the absence of the target epitope and the lack of global expression of specific targets has been shown to evade targeted therapy. Accordingly, in some embodiments of the present invention, gliomas are treated or inhibited by multi-targeted therapies to overcome such limitations. Combinatorial approaches combining IL-13 zetakine with another targeted therapy, such as a chimeric antigen receptor, could overcome the evasion of therapy in tumors that lack the target epitope. This new therapy provided herein has been tested in vitro and in orthotopic xenograft tumor models. In some embodiments, therapies such as IL-13 zetakine-directed immunotherapy are useful on their own against glioblastoma and other IL-13Ra2-positive malignancies or certain subjects with these diseases, or as part of a cure for these diseases or subjects.

Some of the embodiments provided herein include chimeric receptors such as zetakine. In some embodiments, a zetakine comprises a ligand-binding domain, a spacer region, a transmembrane domain, and an intracellular signaling domain (generally comprising a primary signaling domain and a co-stimulatory signaling domain). Some of the embodiments provided herein include second generation IL-13 zetakine, eg, second generation IL-13 zetakine that includes a particular spacer region selected or designed. In some embodiments, the zetakine provided herein includes zetakine with enhanced therapeutic efficacy or in vivo anti-tumor effect or response, or zetakine configured to have enhanced therapeutic efficacy or in vivo anti-tumor effect or response. In some respects, some of the embodiments provided herein are based on the observations set forth herein that specific zetakine in response to target antigens were able to induce the production of more cytokines than reference chimeric receptors that are similar or identical except for the selected spacer region. Also, in some respects, some of the embodiments provided herein are based on the observations described herein that certain zetakine were able to suppress tumor growth over time and significantly increase median survival, such as in orthotopic xenograft tumor models.

DEFINITIONS OF TERMS As used herein , the term "nucleic acid" or "nucleic acid molecule" has its general and ordinary meaning in the light of this specification and includes, but is not limited to, polynucleotides such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonucleolytic or exonucleolytic action. Nucleic acid molecules may be composed of naturally occurring nucleotide monomers (DNA, RNA, etc.), or monomers composed of naturally occurring nucleotide analogs (eg, naturally occurring nucleotide enantiomers), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety or the pyrimidine or purine base moieties. Modifications of sugar moieties include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines or azido groups, and sugar moieties may be etherified or esterified. Furthermore, the entire sugar moiety can be replaced with a conformationally or electronically similar structure, including, for example, an aza-sugar or a carbocyclic sugar analog. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Bonds similar to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoroanilothioate bonds, phosphoranilidate bonds, phosphoramidate bonds, and the like. "Nucleic acid molecule" also includes so-called "peptide nucleic acids," which include natural or modified nucleobases appended to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded. In some embodiments, nucleic acid sequences encoding proteins are provided. In some embodiments, the nucleic acid is RNA or DNA.

As used herein, "vector," "expression vector," or "construct" has its general and ordinary meaning in light of this specification and includes, but is not limited to, nucleic acids that are used to introduce heterologous nucleic acids into cells, which contain various regulatory elements so that the heterologous nucleic acids can be expressed in the cells. Vectors include, but are not limited to, plasmid, minicircle, yeast, or viral genomes. In some embodiments, the vector is a plasmid, minicircle or viral genome. In some embodiments, said vector is a viral vector. In some embodiments, said viral vector is a lentivirus. In some embodiments, said vector is a lentiviral vector. In some embodiments, the vector is a foamy viral vector, an adenoviral vector, a retroviral vector, or a lentiviral vector.

In some embodiments, vectors and sequences that have been modified or optimized, such as by codon optimization, are provided, where codon optimization includes the design process of changing certain codons to codons known to maximize the efficiency of protein expression in desired cells, preferably human cells. In some embodiments, codon optimization is described, and codon optimization using algorithms known to those of skill in the art can generate optimized synthetic gene transcripts for increased protein yield. Programs containing algorithms for codon optimization are known to those skilled in the art. Such programs include, for example, the OptimumGene ^™ algorithm and the GeneGPS® algorithm. In addition, codon-optimized synthetic sequences are commercially available from, for example, Integrated DNA Technologies and other DNA sequencing services. In some embodiments, nucleic acid sequences of genes that encode full-length gene transcripts that are codon-optimized for expression in humans are described. In some embodiments, the gene is specifically optimized to have codons selected to maximize expression of the protein in human cells, such selected codons can increase protein or CAR concentration in T cells.

Codon optimization can also reduce the formation of polynucleotide secondary structures. In some embodiments, codon optimization can also reduce the overall GC/AT ratio. Rigorous codon optimization can result in undesirable secondary structure formation, or secondary structure formation with undesirable GC content. Such secondary structures affect transcription efficiency. By using a program such as GeneOptimizer after codon usage optimization, secondary structure formation can be avoided and GC content can be optimized. Such additional programs can be used to perform further optimizations or troubleshoot after the initial codon optimization, thereby limiting possible secondary structure formation after the first round of optimization. Other programs for optimization are known to those skilled in the art. In some embodiments, the nucleic acid comprises sequences that are codon-optimized for human expression and/or removal of secondary structure and/or reduction of the overall GC/AT ratio. In some embodiments, the sequences are optimized to avoid secondary structure formation. In some embodiments, the sequences are optimized to reduce the overall GC/AT ratio.

Some embodiments include a polypeptide sequence or conservative variations thereof (eg, conservative substitutions of the polypeptide sequence). In some embodiments, "conservative amino acid substitutions" refer to amino acid substitutions in which functionally equivalent amino acids are substituted. Conservative amino acid changes result in synonymous changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids with similar polarities have equivalent functions, resulting in synonymous changes in the amino acid sequence of the resulting peptide. Replacing a particular nonpolar residue with a smaller nonpolar residue may also be considered a "conservative substitution", even if these residues belong to separate groups (e.g., phenylalanine to smaller isoleucine). Amino acid residue families with similar side chains have been defined in the art. Some families involved in conservative amino acid substitutions are shown in Table 1.

The term "chimeric antigen receptor" as used herein has its general and ordinary meaning in light of this specification and includes, but is not limited to, synthetically designed receptors in which the ligand-binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with said disease or disorder is linked via a spacer domain to one or more intracellular signaling domains (e.g. co-stimulatory domains) of a T-cell or other receptor. Chimeric receptors can also be referred to as artificial T-cell receptors, chimeric T-cell receptors, chimeric immune receptors and chimeric antigen receptors (CAR). Monoclonal antibody or binding fragment thereof specificity can be transferred to T cells by transferring coding sequences for chimeric receptors into T cells using vectors such as retroviral and lentiviral vectors. CARs are genetically engineered T cell receptors designed to target T cells to target cells expressing specific cell surface antigens. By a method called adoptive cell transfer, T cells are first obtained from a subject and then genetically engineered to express a receptor capable of exerting specificity for an antigen. The T cells are then reintroduced into the patient. Reintroduced T cells can recognize and target antigens. Such CARs are genetically engineered receptors that can be transplanted with any specificity into cells expressing the immunoreceptor. A chimeric antigen receptor or "CAR" is also understood by some researchers to include an antibody or antibody fragment, a spacer, a signaling domain and a transmembrane region. Because the CARs described herein are genetically engineered in various components or domains (e.g., epitope binding regions (e.g., antibody fragments, scFvs or portions thereof), spacers, transmembrane domains, or signaling domains, etc.) that have resulted in surprising effects, the components of the CAR can often be distinguished as separate entities throughout this disclosure. Different components of the CAR have different variations that, for example, enhance binding affinity for particular epitopes or antigens.

The specificity of a monoclonal antibody or its binding fragment or scFv can be transferred to T cells by using a vector to transfer the CAR coding sequence into the T cells. When using CARs to treat a subject in need thereof, a technique called adoptive cell transfer is used. In this technique, T cells are harvested from a therapeutic subject and the resulting T cells are genetically engineered to express an antigen-specific CAR. The patient is reintroduced with T cells that are capable of recognizing and targeting antigens through CAR expression.

As used herein, the term "ligand" has its general and ordinary meaning in light of this specification and includes, but is not limited to, substances capable of forming complexes with biomolecules. Ligands include, for example, but are not limited to substrates, proteins, small molecules, inhibitors, activators, nucleic acids and neurotransmitters. Ligands bind by intermolecular forces, such as ionic bonds, hydrogen bonds, or van der Waals interactions. When a ligand binds to a receptor protein, it undergoes a three-dimensional conformational change, thereby exerting the ligand's function. The binding strength of a ligand, also called binding affinity, is determined by direct interaction and dissociation. A ligand may be bound by a "ligand binding domain." A "ligand binding domain" may refer, for example, to a conserved sequence found in a structure capable of binding a specific ligand or specific epitope on a protein. A ligand binding domain or ligand binding portion may comprise an antibody or binding fragment or scFv thereof, a receptor ligand or variant thereof, a peptide, and/or a polypeptide affinity molecule or polypeptide binding partner. A ligand binding domain can be, but is not limited to, a particular protein domain or epitope on a protein that is specific for one or more ligands.

As used herein, the term "single-chain variable region fragment" or "scFv" has its general conventional meaning in light of the present specification and includes, but is not limited to, fusion proteins comprising an immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) linked by a short linker peptide. Without limiting the invention in any way, this linker may contain glycine for flexibility, or a hydrophilic amino acid (eg, serine or threonine) for solubility. The linker can connect the N-terminus of VH and the C-terminus of VL, or can connect the C-terminus of VH and the N-terminus of VL. In some embodiments, the ligand binding domain present on the CAR is a single chain variable region fragment (scFV). In some embodiments, the scFv domain present on the CAR is specific for IL-13 receptor α2 (IL13Rα2) present on tumor cells.

In some embodiments, said extracellular domain comprises at least one peptide spacer. In some embodiments, the peptide spacer is 15 amino acids or less in length and 1 amino acid or more or 2 amino acids or more in length. In some embodiments, said spacer is a polypeptide chain. In some embodiments, the length of the polypeptide chain is e.g. length, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 5 4 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, 60 amino acids, 61 amino acids, 62 amino acids, 63 amino acids, 64 amino acids, 65 amino acids, 66 amino acids, 67 amino acids, 68 amino acids, 69 amino acids, 70 amino acids, 71 amino acids, 72 amino acids, 73 amino acids, 74 amino acids, 75 amino acids, 76 amino acids, 77 amino acids, 78 amino acids , 79 amino acids, 80 amino acids, 81 amino acids, 82 amino acids, 83 amino acids, 84 amino acids, 85 amino acids, 86 amino acids, 87 amino acids, 88 amino acids, 89 amino acids, 90 amino acids, 91 amino acids, 92 amino acids, 93 amino acids, 94 amino acids, 95 amino acids, 96 amino acids, 97 amino acids, 98 amino acids, 99 amino acids, 100 amino acids, 101 amino acids, 102 amino acids , 103 amino acids, 104 amino acids, 105 amino acids, 106 amino acids, 107 amino acids, 108 amino acids, 109 amino acids, 110 amino acids, 111 amino acids, 112 amino acids, 113 amino acids, 114 amino acids, 115 amino acids, 116 amino acids, 117 amino acids, 118 amino acids, 119 amino acids, 120 amino acids, 121 amino acids, 122 amino acids, 12 3 amino acids, 124 amino acids, 125 amino acids, 126 amino acids, 127 amino acids, 128 amino acids, 129 amino acids, 130 amino acids, 131 amino acids, 132 amino acids, 133 amino acids, 134 amino acids, 135 amino acids, 136 amino acids, 137 amino acids, 138 amino acids, 139 amino acids, 140 amino acids, 141 amino acids, 142 amino acids, 143 amino acids , 144 amino acids, 145 amino acids, 146 amino acids, 147 amino acids, 148 amino acids, 149 amino acids, 150 amino acids, 151 amino acids, 152 amino acids, 153 amino acids, 154 amino acids, 155 amino acids, 156 amino acids, 157 amino acids, 158 amino acids, 159 amino acids, 160 amino acids, 161 amino acids, 162 amino acids, 163 amino acids, 16 4 amino acids, 165 amino acids, 166 amino acids, 167 amino acids, 168 amino acids, 169 amino acids, 170 amino acids, 171 amino acids, 172 amino acids, 173 amino acids, 174 amino acids, 175 amino acids, 176 amino acids, 177 amino acids, 178 amino acids, 179 amino acids, 180 amino acids, 181 amino acids, 182 amino acids, 183 amino acids, 184 amino acids , 185 amino acids, 186 amino acids, 187 amino acids, 188 amino acids, 189 amino acids, 190 amino acids, 191 amino acids, 192 amino acids, 193 amino acids, 194 amino acids, 195 amino acids, 196 amino acids, 197 amino acids, 198 amino acids, 199 amino acids, 200 amino acids, 201 amino acids, 202 amino acids, 203 amino acids, 204 amino acids, 20 5 amino acids, 206 amino acids, 207 amino acids, 208 amino acids, 209 amino acids, 210 amino acids, 211 amino acids, 212 amino acids, 213 amino acids, 214 amino acids, 215 amino acids, 216 amino acids, 217 amino acids, 218 amino acids, 219 amino acids, 220 amino acids, 221 amino acids, 222 amino acids, 223 amino acids, 224 amino acids, 225 amino acids , 226 amino acids long, 227 amino acids long, 228 amino acids long, 229 amino acids long, 230 amino acids long, 231 amino acids long, 232 amino acids long, 233 amino acids long, 234 amino acids long, 235 amino acids long, 236 amino acids long, 237 amino acids long, 238 amino acids long, 239 amino acids long, or 240 amino acids long, or a length within a range defined by any two of these lengths. In a chimeric receptor, the spacer may comprise, for example, any of the 20 amino acids in any order to form a polypeptide chain of desired length, wherein the 20 amino acids include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, or tryptophan. In some embodiments, a spacer is located between the scFV or ligand binding domain and the transmembrane region in the chimeric receptor. By customizing, selecting, configuring or optimizing the spacer to a desired length, binding of the scFv or ligand binding domain to target cells may be improved, thereby increasing cytotoxicity. In some embodiments, the linker or spacer located between the scFv domain or ligand binding domain and the transmembrane domain can be 25 to 55 amino acids long (e.g., at least 25 amino acids long, at least 26 amino acids long, at least 27 amino acids long, at least 28 amino acids long, at least 29 amino acids long, at least 30 amino acids long, at least 31 amino acids long, at least 32 amino acids long, at least 33 amino acids long, at least 34 amino acids long, at least 35 amino acids long, at least 36 amino acids long, at least 37 amino acids long, at least 38 amino acids long). Amino acids in length, at least 39 amino acids in length, at least 40 amino acids in length, at least 41 amino acids in length, at least 42 amino acids in length, at least 43 amino acids in length, at least 44 amino acids in length, at least 45 amino acids in length, at least 46 amino acids in length, at least 47 amino acids in length, at least 48 amino acids in length, at least 49 amino acids in length, at least 50 amino acids in length, at least 51 amino acids in length, at least 52 amino acids in length, at least 53 amino acids in length, at least 54 amino acids in length, or at least 55 amino acids in length, or within a range defined by any two of these lengths may be used). Examples of spacers include spacers consisting of only the IgG4 hinge, spacers consisting of the IgG4 hinge linked to the CH2 and CH3 domains, or spacers consisting of the IgG4 hinge linked to the CH3 domain. Examples of spacers also include spacers described in Hudecek et al. Clin. Cancer Res., 19:3153 (2013), International Patent Application WO2014031687, U.S. Pat.

In some embodiments, a CAR signaling domain, such as a first signaling domain or a costimulatory signaling domain, includes an intracellular or cytoplasmic domain of a protein or receptor protein that interacts with an intracellular component, which intracellular domain can relay a signal or participate in the relaying of a signal. In some embodiments, such interactions can occur through signaling by the intracellular domain via specific protein-protein or protein-ligand interactions with the effector molecule or effector protein, which then undergoes a signaling chain reaction to send the signal to its destination. In some embodiments, signaling domains include co-stimulatory domains. In some embodiments, co-stimulatory domains include signaling moieties that provide signals to T cells that, in addition to the primary signal provided, for example, by the CD3 zeta chain of the TCR/CD3 complex, provide signals to the T cells that enhance responses such as, for example, T cell effector responses such as immune response, activation, proliferation, differentiation, cytokine secretion, cytolytic activity, perforin activity, or granzyme activity. In some embodiments, the intracellular signaling domain and/or costimulatory domain can include, but is not limited to, all or part of CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3, or all or part of a ligand that specifically binds to CD83.

In some embodiments, the compositions, cells and vectors of the invention comprise a marker sequence or nucleic acid encoding the marker sequence, which can include, for example, proteins that label cells. In some of the cell embodiments of the invention, the cell of the invention co-expresses a marker protein for the particular chimeric protein expressed on the cell. In some of the cell embodiments provided herein, the chimeric receptor is co-expressed with a particular marker protein. In some of the cell embodiments provided herein, the cell comprises a nucleic acid encoding a chimeric receptor. Markers can include selectable marker sequences and include, for example, genes that are introduced into vectors or cells to confer a property that allows them to be selected artificially. The selectable marker sequence or marker sequence may be a screenable marker, and the use of such marker sequences allows researchers to distinguish between desired and undesired cells, or to enrich for a particular type of cell. In some embodiments, chimeric receptor-encoding vectors are provided that include a marker sequence that encodes a cell surface selectable marker. In embodiments described herein, CARs are provided that may contain marker sequences that are selectable in experiments such as flow cytometry. In some embodiments, the marker is Her2tG protein or EGFRt protein.

As used herein, "methotrexate (MTX)" has its general ordinary meaning in light of this specification and includes, but is not limited to, antimetabolites and antifolates. Methotrexate (MTX) works by inhibiting folic acid metabolism. In some embodiments, methods are provided for generating recombinant multiplexed T cells for adoptive T cell immunotherapy. In its broadest scope, the method of the invention may comprise providing a gene delivery polynucleotide according to any of the embodiments described herein and selecting for cells containing said gene delivery polynucleotide by adding a selection reagent. In some of the embodiments described herein, the selection reagent comprises a selection agent. In some embodiments, the selection reagent is MTX.

The term "dihydrofolate reductase" or "DHFR" as used herein has its general and ordinary meaning in light of the specification, including, but not limited to, an enzyme that uses NADPH as an electron donor to reduce dihydrofolate to tetrahydrofolate, which can be converted to a tetrahydrofolate-derived cofactor used in the C1 transfer reaction. In some of the embodiments herein, gene delivery polynucleotides are provided. In some embodiments, the gene delivery polynucleotide comprises at least one selectable marker cassette encoding a double mutant of dihydrofolate reductase (DHFRdm).

As used herein, a "ribosome skip sequence" refers to a sequence that has the function of causing the translating ribosome to "skip" the ribosome skip sequence, preventing peptide bond formation and translation from the region immediately following the ribosome skip sequence. For example, some viruses have ribosomal skipping sequences that allow them to sequentially translate multiple proteins from a single nucleic acid, resulting in the translated proteins being separate proteins that are not linked by peptide bonds. Ribosome skipping sequences are used herein as "linker" sequences. In some of the nucleic acid embodiments provided herein, the nucleic acid of the invention includes a ribosome skip sequence between the chimeric receptor-encoding sequence and the marker protein-encoding sequence, such that the chimeric receptor and marker protein are co-expressed without being linked by a peptide bond. In some embodiments, said ribosome skip sequence is a P2A sequence, T2A sequence, E2A sequence or F2A sequence. In some embodiments, said ribosome skip sequence is a T2A sequence.

As used herein, "zetakine" may refer to a specific type of CAR that contains a cytokine in its ligand-binding domain that is capable of specifically binding to a cytokine receptor. In some embodiments, such cytokines include human cytokines, such as IL-13. In some embodiments, the IL-13 contains mutations in sequence and exhibits increased affinity for IL-13 receptor α2.

As used herein, "mutein" has its general and ordinary meaning in light of the specification and includes, but is not limited to, proteins resulting from mutation. A "IL-13 mutein" may be an IL-13 variant that may have at least one or at least two amino acid substitutions.

As used herein, the term "interleukin 13 receptor α2 subunit (IL-13Rα2)," also known as CD213A2 (cluster of differentiation 213A2), has its general and ordinary meaning in light of the specification, and includes, for example, but is not limited to, the cell membrane-associated protein encoded by the IL-13RA2 gene in humans. IL-13Rα2 is closely related to IL-13Rα1, one of the subunits of the interleukin-13 receptor complex. IL-13Rα2 normally binds IL-13 with high affinity, but has no functional cytoplasmic domain and does not appear to function as a mediator of signaling. On the other hand, IL-13Rα2 can regulate the actions of IL-13 and IL-4, although it cannot bind to IL-4 directly. Furthermore, IL-13Rα2 has been reported to play a role in the internalization of IL-13.

As used herein, "T cells" or "T lymphocytes" may be T cells obtained from any mammalian species, such as monkeys, dogs, humans, etc., but are preferably T cells obtained from primates. In some embodiments, the T cells are allogeneic (allogeneic but derived from another donor) to the recipient subject to which the T cells will be administered or to which the T cells have been administered, e.g., in the form of a therapeutic composition. In some embodiments, the T cells are autologous (donor and recipient are the same). In some embodiments, the T cells are syngeneic (donor and recipient are different but identical twins).

As used herein, the term "cytotoxic T lymphocytes (CTL)" has its general and ordinary meaning in light of the specification and includes, but is not limited to, T lymphocytes that express CD8 on their surface (e.g., CD8 ⁺ T cells). In some embodiments, such cells are preferably antigen-experienced “memory” T cells ( _TM cells). In some embodiments, said cells are cytotoxic T lymphocytes. As used herein, a "central memory" T cell (or " _TCM ") is an antigen-experienced cytotoxic T lymphocyte (CTL) that expresses CD62L, CCR-7 and/or CD45RO on its surface but does not express CD45RA or has reduced expression of CD45RA compared to naive cells. In some embodiments, the cells are central memory T cells (T _CM ). In some embodiments, the central memory cells may have positive expression of CD62L, CCR7, CD28, CD127, CD45RO or CD95, or a combination thereof, and reduced expression of CD54RA compared to naive cells. As used herein, "effector memory" T cells (or " _TEM ") are antigen-experienced T cells that express no or reduced CD62L on their surface compared to central memory cells and no or reduced CD45RA expression compared to naive cells. In some embodiments, said cells are effector memory T cells. In some embodiments, effector memory cells may be negative for CD62L and/or CCR7 expression and positive or negative for CD28 and/or CD45RA expression compared to naive or central memory cells.

Mature T cells express CD4 as a surface protein and are called CD4+ T cells. CD4+ T cells are generally treated as destined to function as helper T cells in the immune system. For example, when an antigen-presenting cell expresses antigen on class II MHC, CD4+ cells assist the antigen-presenting cell through a combination of cell-cell interactions (eg, CD40 and CD40L) and cytokines. However, there are rare exceptions, for example subgroups of regulatory T cells, natural killer cells or cytotoxic T cells also express CD4. None of these CD4+ expressing T cell populations are considered helper T cells.

The term "central memory" T cells (or " _TCM ") as used herein has its general and ordinary meaning in light of the present specification, e.g., antigen-experienced cytotoxic T lymphocytes (CTLs) that express CD62L, CCR-7 or CD45RO, or any combination thereof, on their surface compared to naive cells, but do not express CD45RA or have reduced expression of CD45RA (CTLs). ), but is not limited thereto. In some embodiments, the central memory cells have positive expression of CD62L, CCR7, CD28, CD127, CD45RO or CD95, or any combination thereof, and reduced expression of CD54RA compared to naive cells.

The term “effector memory” T cells (or “T _EM ”) as used herein, in light of the present specification, has its general and ordinary meaning, including, but not limited to, antigen-experienced T cells that have no or reduced CD62L expression on their surface compared to central memory cells and no or reduced CD45RA expression compared to naive cells. not. In some embodiments, effector memory cells are negative for expression of CD62L or CCR7 or both and positive or negative for expression of CD28 or CD45RA or both compared to naive cells or central memory cells.

The term "naive" T cells as used herein, in light of the present specification, has its general and ordinary meaning, for example, T lymphocytes that have not experienced antigen, including but not limited to T lymphocytes that express CD62L or CD45RA, or both, and do not express CD45RO, as compared to central or effector memory cells. In some embodiments, the naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers, where the naive T cell phenotypic markers include CD62L, CCR7, CD28, CD127 or CD45RA, or any combination thereof.

As used herein, the term "effector" T cell or "T _E " has its general conventional meaning in light of the present specification, e.g., an antigen-experienced cytotoxic T lymphocyte that does not express CD62L, CCR7 or CD28, has reduced expression of CD62L, CCR7 or CD28, or is positive for granzyme B or perforin or a combination thereof compared to central memory cells or naive T cells. It includes, but is not limited to, T lymphocytes.

As used herein, the term "progenitor T cell" in light of this specification has its general and ordinary meaning, including, but not limited to, lymphoid progenitor cells that are capable of migrating to the thymus and becoming progenitor T cells and that do not express T cell receptors. All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells (lymphoid progenitor cells) derived from hematopoietic stem cells colonize the thymus, proliferate by cell division, and produce a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classified as double-negative (CD4 ⁻ CD8 ⁻ ) cells. As they develop, they become double-positive thymocytes (CD4 ⁺ CD8 ⁺ ) and finally mature into single-positive (CD4 ⁺ CD8 ⁻ or CD4 ⁻ CD8 ⁺ ) thymocytes, which are then released from the thymus into peripheral tissues.

The terms "pharmaceutical excipient" or "pharmaceutical carrier" as used herein have their general and ordinary meaning in light of the specification and include, but are not limited to, carriers or inert media used as solvents for formulating and/or administering pharmaceutically active agents or T cells for treatment or therapeutic purposes. Carriers include polymeric micelles, liposomes, lipoprotein-based carriers, nanoparticle carriers, dendrimers or other carriers known to those skilled in the art for T cells. An ideal carrier or excipient would be non-toxic, biocompatible, non-immunogenic, biodegradable, not recognized by host defense mechanisms, or any combination of such properties.

As used herein, "subject" or "patient" refers to any organism to which the embodiments described herein may be used or administered, eg, for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Subjects or patients include, for example, animals. In some embodiments, the subject is a mouse, rat, rabbit, non-human primate or human. In some embodiments, the subject is bovine, ovine, porcine, equine, canine, feline, primate, or human.

It has been found that interleukin 13 receptor α2 (IL-13Rα2) is highly expressed in various cancer cells such as pancreatic cancer, breast cancer, ovarian cancer, and malignant glioma (eg, glioblastoma). IL-13Rα2 may also be overexpressed in a majority of human patients with high-grade astrocytomas (see PLoS One. 2013 Oct 16; 8(10):e77719; which is expressly incorporated herein by reference in its entirety). Furthermore, suppression of IL13RA2 expression levels in various models of cancer cells can significantly retard tumor growth (Breast Cancer Research, 2015; 17 (1); this document is expressly incorporated herein by reference in its entirety). Most normal tissues do not express IL-13RA2, and in such cases IL-13RA2 would be expected to be expressed at low levels. Also, in the case of glioblastoma multiforme (GBM), high expression of IL13Rα2 may be a prognostic marker of advanced tumors and poor patient survival.

Some of the embodiments provided herein include chimeric receptors (such as zetakine), such as chimeric receptors directed against solid tumors. In some embodiments, the extracellular domain comprises a binding domain comprising a variant of IL13 (such as IL13 E13Y), which is linked via a spacer region to the transmembrane domain of the chimeric receptor. In some embodiments, this spacer region generally extends from the binding domain to the transmembrane domain. In some embodiments, the spacer comprises a polypeptide that is 12 amino acids long, 15 amino acids long, 20 amino acids long, 30 amino acids long, 40 amino acids long, 50 amino acids long, 60 amino acids long, 70 amino acids long, 80 amino acids long, 90 amino acids long, or greater than 100 amino acids long; comprises a polypeptide that is 0 amino acids long, about 80 amino acids long, about 90 amino acids long, or greater than about 100 amino acids long; including polypeptides that are 20 amino acids in length, at least about 30 amino acids in length, at least about 40 amino acids in length, at least about 50 amino acids in length, at least about 60 amino acids in length, at least about 70 amino acids in length, at least about 80 amino acids in length, at least about 90 amino acids in length, or at least about 100 amino acids in length; , 50-150 amino acids in length, or 110 amino acids in length or about 110 amino acids in length. In some embodiments, the transmembrane domain is or comprises a CD28 transmembrane domain (CD28tm), which is followed by a co-stimulatory domain, which co-stimulatory domain is, for example, a co-stimulatory domain derived from the intracellular segment of human 4-1BB (CD137) and a first signaling domain, such as the signaling domain of CD3zeta.

In some embodiments, the chimeric receptor-encoding nucleic acid further comprises a marker-encoding sequence, which may be operably linked to the same promoter to which the chimeric receptor-encoding nucleic acid is linked. In some embodiments, the marker is a truncated form of a cell surface receptor, such as epidermal growth factor receptor (EGFRt), CD19 (CD19t) or HER2 (Her2t) or other receptors. In some embodiments, the marker-encoding nucleic acid is separated from the chimeric receptor-encoding nucleic acid by a skip sequence, such as a P2A ribosome skip sequence, a T2A ribosome skip sequence, or an IRES. In some embodiments, the nucleic acid further comprises a transgene encoding a double mutant of dihydrofolate reductase (DHFRdm), which may be added, for example, to facilitate selection of cells expressing the construct product (such as therapeutic T cell products) with methotrexate.

Specific Nucleic Acids Some of the embodiments of the methods and compositions provided herein comprise nucleic acids encoding zetakine, a receptor targeted by a cell membrane-bound IL-13 mutein. In some embodiments, the nucleic acid is
a) a first polynucleotide encoding an extracellular domain;
b) a second polynucleotide encoding a mutein of IL-13;
c) a third polynucleotide encoding a transmembrane domain; and d) a fourth polynucleotide encoding an intracellular signaling domain.
In some embodiments, the nucleic acid further comprises a polynucleotide encoding a marker polypeptide (eg, EGFRt, etc.). In some embodiments, the nucleic acid further comprises a polynucleotide encoding a selectable marker (eg, DHFRdm, etc.). In some embodiments, the nucleic acid comprises a ribosome skip sequence.

In some embodiments, the mutein of IL13 comprises, consists of, or consists essentially of an amino acid sequence having (%) identity with the amino acid sequence of SEQ ID NO:16. In some of such embodiments, the sequence identity to SEQ ID NO: 16 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, or a percentage between any two of these percentages. In some embodiments, the IL13 mutein comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:16.

In some embodiments, said extracellular domain comprises at least one peptide spacer. In some embodiments, the peptide spacer is 15 amino acids or less in length and 1 amino acid or more or 2 amino acids or more in length. In some embodiments, said spacer is a polypeptide chain. In some embodiments, said spacer comprises an IgG4 hinge spacer or portion thereof. In some embodiments, the spacer comprises the hinge region or portion thereof of a human antibody. In some embodiments of the method, the spacer comprises the hinge region of IgG4 or a portion thereof. In some embodiments, said IgG4 hinge region is a modified IgG4 hinge or portion thereof. A "modified IgG4 hinge" as described herein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with the amino acid sequence of the hinge region set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or any percentage of these It may also refer to a hinge region that may have sequence identity within the range defined by any two. In some embodiments, the spacer is a short (S) spacer, a medium length (M) spacer or a long (L) spacer. A short (S) spacer comprises the sequence shown in SEQ ID NO:9. A medium length (M) spacer comprises the sequence shown in SEQ ID NO:10. A long (L) spacer comprises the sequence shown in SEQ ID NO:11.

In some embodiments, the spacer comprises, consists of, or consists essentially of an IgG4 hinge spacer (short (S) spacer), an IgG4 hinge-CH3 spacer (medium length (M) spacer), or an IgG4 hinge-CH2-CH3 spacer. In some embodiments, the spacer comprises an IgG4-CH3 spacer (medium length (M) spacer).

In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having a % sequence identity with the amino acid sequence of SEQ ID NO:10. In some of such embodiments, the sequence identity to SEQ ID NO: 10 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, or a percentage between any two of these percentages. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:10.

In some embodiments, the spacer comprises, consists of, or consists essentially of an amino acid sequence having a % sequence identity with the amino acid sequence of SEQ ID NO:11. In some of such embodiments, the sequence identity to SEQ ID NO: 11 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, or a percentage between any two of these percentages. In some embodiments, the spacer comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:11.

In some embodiments, the spacer is at least 10-229 amino acids, at least 10-200 amino acids, at least 10-175 amino acids, at least 10-150 amino acids, at least 10-125 amino acids, at least 10-100 amino acids, at least 10-75 amino acids, at least 10-50 amino acids, at least 10-40 amino acids, at least 10-30 amino acids, at least 10-30 amino acids, It has 20 amino acids, or at least 10-15 amino acids, or any integer number of amino acids between the extremes of these ranges. In some embodiments, the spacer region has 1-12 amino acids, 1-119 amino acids, or 1-229 amino acids. In some embodiments, the spacer is 1 to less than 250 amino acids long, 1 to less than 200 amino acids long, 1 to less than 150 amino acids long, 1 to less than 100 amino acids long, 1 to less than 75 amino acids long, 1 to less than 50 amino acids long, 1 to less than 25 amino acids long, 1 to less than 20 amino acids long, 1 to less than 15 amino acids long, 1 to less than 12 amino acids long. Less than an amino acid in length, or greater than or equal to 1 amino acid and less than 10 amino acids in length. In some embodiments, the spacer is 10-250 amino acids long, 10-150 amino acids long, 10-100 amino acids long, 10-50 amino acids long, 10-25 amino acids long, 10-15 amino acids long, 15-250 amino acids long, 15-150 amino acids long, 15-100 amino acids long, 15-50 amino acids long, 15-25 amino acids long, 25-250 amino acids long, 25-100 amino acids long. long, 25-50 amino acids long, 50-250 amino acids long, 50-150 amino acids long, 50-100 amino acids long, 100-250 amino acids long, 100-150 amino acids long, or 150-250 amino acids long. Exemplary spacers include a spacer consisting of the IgG4 hinge alone, a spacer consisting of the IgG4 hinge linked to the CH2 and CH3 domains, or a spacer consisting of the IgG4 hinge linked to the CH3 domain. Representative spacers also include, but are not limited to, spacers described in Hudecek et al. Clin. Cancer Res., 19:3153 (2013), International Patent Application WO2014031687, U.S. Patent No. 8,822,647, U.S. Published Application No. 2014/0271635, which are expressly incorporated herein by reference in their entireties.

In some embodiments, a "transmembrane domain" is a hydrophobic protein region that spans the cell membrane bilayer and serves to anchor proteins embedded in biological membranes. The topology of the transmembrane domain may be, but is not limited to, a transmembrane type α-helix. In some embodiments, the transmembrane domain comprises a CD28 transmembrane sequence or fragment thereof, wherein the CD28 transmembrane sequence or fragment thereof is, e.g. , 27 amino acids long or 28 amino acids long, or any length within the range defined by any two of these lengths. In some embodiments, the CD28 transmembrane sequence or fragment thereof is 28 amino acids long.

In some embodiments, the intracellular signaling domain comprises a costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and combinations thereof, in combination with all or part of the CD3ζ domain. In some embodiments, the intracellular signaling domain comprises the signaling functional portion of the CD3ζ domain and the co-stimulatory functional portion of the 4-1BB domain.

In some embodiments, the nucleic acid further comprises a sequence encoding a marker sequence. In some embodiments, the marker is a truncated form of a cell surface receptor, such as epidermal growth factor receptor (EGFRt), CD19 (CD19t) or HER2 (Her2t) or other receptors. In some embodiments, the marker sequence is a truncated form of a cell surface receptor, which may be EGFRt.

In some embodiments, the nucleic acid further comprises a transgene encoding dihydrofolate reductase configured to be selectable by methotrexate. In some embodiments, the transgene encoding dihydrofolate reductase is a dihydrofolate reductase double mutant (DHFRdm). In some embodiments, the dihydrofolate reductase double mutant comprises an L22F amino acid mutation and an F31S amino acid mutation.

In some embodiments, the nucleic acid further comprises a sequence encoding a ribosome skip sequence. In some embodiments, said ribosome skip sequence comprises P2A or T2A.

In some embodiments, the nucleic acid has been modified such that the overall nucleic acid has a reduced GC/AT ratio. In some embodiments, the nucleic acid is codon-optimized for expression in humans.

Some of the embodiments of methods and compositions provided herein comprise an expression vector comprising a nucleic acid according to any of the embodiments of the invention. In some embodiments, said vector is a viral vector. In some embodiments, the vector is a lentiviral or adenoviral vector. In some embodiments, said receptor zetakine comprises an extracellular domain comprising an IL-13 mutein and a spacer; a transmembrane domain; and an intracellular signaling region, wherein said spacer is located between said mutein and said transmembrane domain.

Certain Chimeric Receptors Some of the embodiments of the methods and compositions provided herein comprise a chimeric receptor polypeptide encoded by a nucleic acid according to any of the embodiments of the invention or a vector according to any of the embodiments of the invention. Some of the embodiments of the methods and compositions provided herein comprise zetakine, a receptor targeted by a cell membrane-bound IL13 mutein encoded by a nucleic acid according to any of the embodiments of the invention or a vector according to any of the embodiments of the invention.

Specific Host Cells Some of the embodiments of the methods and compositions provided herein comprise host cells containing nucleic acids according to any of the embodiments of the invention or expression vectors according to any of the embodiments of the invention. In some embodiments, said host cells comprise genetically modified cells. In some embodiments, said host cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some embodiments, said CD8+ cytotoxic T lymphocytes are central memory T cells, and said central memory T cells are CD45RO+, CD62L+ and CD8+. In some embodiments, said host cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells and bulk CD4+ T cells. In some embodiments, said CD4+ helper lymphocytes are naive CD4+ T cells, and said naive CD4+ T cells are CD45RA+, CD62L+ and CD4+, and CD45RO-. In some embodiments, said host cell is a progenitor T cell. In some embodiments, said host cells are hematopoietic stem cells.

Some of the method and composition embodiments provided herein provide host cells according to any of the embodiments of the invention or compositions according to any of the embodiments of the invention for use in the treatment or suppression of cancers or solid tumors that express IL-13 receptor α2. The composition comprises host cells and excipients according to any of the embodiments of the invention. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL13Rα-positive malignancy. In some embodiments, the cancer is brain cancer or brain tumor. Accordingly, some embodiments relate to host cells according to any of the embodiments described herein for use in medicine or in the treatment or control of cancers, such as brain cancer, including but not limited to IL13Rα-positive malignancies, glioblastoma multiforme (GBM), gliomas and glioblastomas.

Certain Compositions Some of the embodiments of methods and compositions provided herein comprise compositions comprising host cells according to any of the embodiments of the invention and pharmaceutically acceptable excipients.

Certain Methods of Making Host Cells Some of the embodiments of methods and compositions provided herein include methods of making host cells according to any of the embodiments of the present invention. Some of such embodiments are
a) introducing a nucleic acid according to any embodiment of the invention or an expression vector according to any embodiment of the invention into a lymphocyte;
b) culturing said lymphocytes in the presence of an anti-CD3 or anti-CD28 antibody and at least one homeostatic cytokine; and c) selecting said lymphocytes with a selection reagent configured to selectively enrich for cells transduced with said nucleic acid or said vector.
In some embodiments, the agent of choice is methotrexate. In some embodiments, the lymphocytes have a CD45RA-, CD45RO+ or CD62L+ phenotype, or any combination thereof. In some embodiments, said lymphocytes are CD8+ or CD4+. In some embodiments, said cytokine is IL-15, IL-7 or IL-21 or any combination thereof. In some embodiments, the method further comprises introducing a second nucleic acid encoding a marker protein into the host cell. In some embodiments, said marker protein is EGFRt. In some embodiments, said expression vector comprises a nucleic acid according to any of the embodiments of the invention. In some embodiments, said vector is a viral vector. In some embodiments, the vector is a lentiviral or adenoviral vector.

In some embodiments, said host cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some embodiments, said CD8+ cytotoxic T lymphocytes are central memory T cells, and said central memory T cells are CD45RO+, CD62L+ or CD8+ or any combination thereof. In some embodiments, said host cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells and bulk CD4+ T cells. In some embodiments, said CD4+ helper lymphocytes are naive CD4+ T cells, and said naive CD4+ T cells are CD45RA+, CD62L+ or CD4+ or any combination thereof, and are CD45RO-. In some embodiments, said host cell is a progenitor T cell. In some embodiments, said host cells are hematopoietic stem cells.

Certain therapeutic methods Some of the embodiments of the methods and compositions provided herein involve the use of the host cells of the invention in therapeutic methods. Some of such embodiments involve the use of cells in the treatment, suppression or amelioration of cancers or solid tumors expressing IL-13 receptor alpha2 (IL-13Ra2). In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is IL-13Rα positive malignancy. In some embodiments, the cancer is brain cancer or brain tumor.

Some of the method and composition embodiments provided herein provide a method of performing cellular immunotherapy in a subject having a cancer or tumor, comprising administering to said subject a host cell according to any embodiment of the invention or a composition according to an embodiment of the invention. The composition comprises host cells according to any of the embodiments of the invention and pharmaceutically acceptable excipients. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is an IL13Rα-positive malignancy. In some embodiments, the cancer is brain cancer. In some embodiments, the subject is a subject selected for administration of a combination therapy. In some embodiments, said combination therapy comprises administration of a chemotherapeutic agent. In some embodiments, said combination therapy comprises administration of radiation therapy. In some embodiments, the chemotherapeutic agent is electrochemotherapy, an alkylating agent, an antimetabolite (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexine). sart, pemetrexed (Alimta®, pentostatin or thioguanine), anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators or checkpoint inhibitors (checkpoint kinase CHK1 or CHK2). In some embodiments, the cancer is glioma.

Example 1 Construction of IL-13(E13Y) Zetakine CARs Various IL-13(E13Y) zetakine CARs with different spacer regions were constructed. As shown in FIG. 1, the IL-13 (E13Y) zetakine CAR-encoding nucleic acid comprises a leader sequence (EF1p); a polynucleotide encoding an IL-13 mutein (E13Y); a polynucleotide encoding any one of three spacers; a polynucleotide encoding a CD28tm transmembrane sequence; I was there. The three spacers were a short 'S' spacer containing a modified IgG4 hinge; a medium-length 'M' spacer containing a modified IgG4 hinge and an immunoglobulin CH3 region (IgG4 hinge-CH3); and a long 'L' spacer containing a modified IgG4 hinge and an immunoglobulin CH2 domain and an immunoglobulin CH3 domain (IgG4 hinge-CH2-CH3). The L-spacer contains two mutations (L235D, N297Q) in the CH2 domain that can reduce the likelihood of CAR interacting with the Fc receptor (FcR) to produce undesirable effects. In addition, CAR contained a self-cleaving ribosomal skipping sequence 2A peptide (T2A). In some constructs, the lentivector further contains a transgene encoding a double mutant of dihydrofolate reductase (DHFRdm) configured to be selectable by methotrexate. A mutein of IL-13 is shown in SEQ ID NO:16. An exemplary IL-13 mutein sequence is described in Kahlon et al. (Kahlon KS et al., Cancer Res. 2004; which is incorporated herein by reference in its entirety). As described herein, the E13Y mutation improved selective binding to IL-13Rα2.

Example 2 - In vitro comparison of various CARs with different spacers T cells with different CARs were generated according to the method shown in Figure 2A. First, CD8+ T cells were selected from peripheral blood mononuclear cells (PBMC) using microbeads. After stimulation of the selected cells with CD3/CD28 microbeads, lentivirus was used to transduce the construct. In several replicate experiments (*), the CD8 T cells were transduced with a construct further comprising T2A-DHFRdm inserted in-frame downstream of EGFRt to co-express T2A-DHFRdm, allowing selection of MTX-resistant T cells co-expressing IL13 zetakine CAR in functionally relevant amounts. Cells were then stained by flow cytometry to identify cells expressing zetakine CAR, and the EGFRt marker was used to select cells. Representative flow cytometry data shown in FIG. 2B showed that CD8+ T cells were efficiently selected and then expression of a surrogate marker (EGFRt) could be exploited to enrich or select nearly pure IL13 zetakine CAR-expressing CD8+ T cells (*).

In addition, the specific lysis of IL13Ra2-expressing target cells by CD8+ T cells containing zetakine CARs with S, M or L spacers and the expression of cytokines by the CD8+ T cells in the presence of target cells was assessed. First, we analyzed the expression of IL13Ra2 in U87, U251T and DAOY cells as target cells (Fig. 3A). For the 4-hour ⁵¹ Cr cytotoxicity assay, labeled U87, U251T and DAOY tumor cell lines were co-cultured with Mock T cells or various second-generation IL13 zetakine CAR T cells with different spacers (Fig. 3B). Specific lysis was induced in IL13 zetakine CAR T cells with an M spacer and IL13 zetakine CAR T cells with an L spacer, whereas IL13 zetakine CAR T cells with a short spacer failed to target IL13Ra2 efficiently. Data represent the mean±SD of 3 different donors.

For cytokine release assays, T cells containing zetakine CAR were cultured with target cells for 24 hours. Cell-free supernatants were harvested and TNFα, IFNγ and IL-2 secretion were measured. CD8 T cells containing zetakine CARs with M spacers and CD8 T cells containing zetakine CARs with L spacers produced high amounts of cytokines, whereas CD8 T cells containing zetakine CARs with S spacers produced little cytokine (Fig. 3C).

Example 3 - In Vivo Antitumor Activity of IL13 Zetakine CARs The in vivo antitumor activity of IL13 zetakine CARs containing M or L spacers was tested. On day 0, ffLuc-labeled U87 glioblastoma cells (0.2×10 ⁶ ) were injected intracranially into the forebrain of NSG mice. On day 7, mice (n=5 per group) were administered CD8+ T cells transduced with medium- or long-spacer second-generation IL13 zetakine CARs at varying doses (2× ¹⁰ or 1× ¹⁰ ), or Mock T cells were administered to mice.

Total flux (photons/second) from U87 cells was measured as an index of tumor burden. As shown in FIG. 4A, tumor burden decreased over time in mice treated with cells containing IL13 zetakine CARs containing L spacers compared to Mock-treated mice and mice treated with IL13 zetakine CARs containing L spacers. Kaplan-Meier survival curves showed improved survival in mice treated with various doses of cells containing the IL13 zetakine CAR containing the L spacer (Fig. 4B).

Example 4 In Vitro Analysis of IL13 Zetakine CAR Cells containing IL13 zetakine CAR with M or L spacers were generated in substantially the same manner as shown in FIG. 5A. Briefly, CD4+ T cells and CD8+ T cells were isolated and co-cultured. Two days later, these T cells were transduced with IL-13 zetakine with an M spacer or IL-13 zetakine with an L spacer. After termination of stimulation (S1D13), cells were analyzed for CD4:CD8 ratio, expression of markers and direct expression of zetakine by staining with anti-IL-13 antibody (Fig. 5B).

Cells were tested in an in vitro functional assay (Fig. 6). After co-culture of IL-13 zetakine-targeted T cells with IL-13Ra2-expressing target cells, a chromium release assay was performed to assess the extent of specific lysis. In this assay, stimulated day 15 (S1D15) T cells were co-cultured with different target cell lines, with varying ratios of effector cells to target cells. K562-OKT3 cells were used as a positive control for activation of T cells by induction of the TCR complex, and other cell lines expressed IL-13Ra2 to varying degrees (see table above). The bottom table shows the ratio of CD4 and CD8 in T cells and the positive rate of surrogate markers. Assays were performed by normalizing the total number of surrogate marker-positive cells. The results showed that both zetakine with an M spacer and zetakine with an L spacer induced lysis of IL-13Ra2-positive target cells.

Example 5 In Vivo Anti-Tumor Activity of IL13 Zetakine CAR As shown in Figure 7, IL-13 zetakine with an M spacer and IL-13 zetakine with an L spacer were observed to suppress tumor growth in a glioblastoma (GBM) model. Cells generated in Example 4 and stimulated day 15 (S1D15) cells were tested in an orthotopic xenograft tumor model. This model was generated by intracranial injection of GFP:ffluc-expressing U87 glioblastoma cells into NOD-Scid IL2yR-null mice. Seven days later, IL-13 zetakine-targeted T cells were injected ipsilaterally with GFP:ffluc-expressing U87 tumors. Evaluation of the flux generated from ffluc-expressing U87 tumors showed that IL-13 zetakine-expressing T cells with M or L spacers reduced tumor burden in vivo, indicating that these IL-13 zetakine-expressing T cells suppressed tumor growth (Fig. 7). Statistical analysis of changes in flux 26 days after tumor inoculation showed that IL-13 zetakine-expressing T cells with M or L spacers significantly suppressed flux development compared to Mock T cells (without IL-13 zetakine).

Example 6 - In Vitro Analysis of IL13 Zetakine CAR
IL-13 zetakine-targeted T cells were analyzed in a chromium release assay. In this assay, rapid cultured day 14 (S1R1D14) T cells were co-cultured with different target cell lines, with varying effector to target cell ratios. K562-OKT3 cells were used as a positive control for activation of T cells by induction of the TCR complex, and other cell lines expressed IL-13Ra2 to varying degrees (see table above). To demonstrate IL-13Ra2-specific lysis, Mock T cells not transduced for zetakine expression were included in this study as a negative control. The bottom table shows the ratio of CD4 and CD8 in T cells and the positive rate of surrogate markers. Assays were performed by normalizing the total number of marker-positive cells. Results showed that both zetakine with an M spacer and zetakine with an L spacer induced lysis of IL-13Ra2-positive target cells (FIG. 8).

In another study, IL-13 zetakine-expressing T cells were co-cultured with various target cell lines for 24 hours. Here, the ratio of effector cells to target cells was 2:1. The test data indicated that the tested zetakine was able to target-specifically bind to IL-13Ra2 and induce cytokine release (Figure 9).

A listing of specific amino acid and nucleotide sequences in the embodiments provided herein is provided in Table 2.

As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by," and is intended to be open-ended and inclusive, and does not exclude additional elements or steps not described herein.

The foregoing description discloses several methods and materials of the present invention. The methods and materials of the invention may vary, as may manufacturing methods and equipment. Such modifications can be readily understood by one of ordinary skill in the art in light of the practice of the invention disclosed herein or in light of the present disclosure. This invention, therefore, is not limited to the particular embodiments disclosed herein, but encompasses all modifications and other aspects possible within the true scope and spirit of the invention.

All references, including but not limited to published patent applications, unpublished patent applications, patents and scientific literature, cited herein are hereby incorporated by reference in their entirety and made a part hereof. To the extent any document, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure herein controls and/or supersedes any such conflict.

Claims (47)

A nucleic acid encoding zetakine, a receptor targeted by an IL-13 mutein,
the receptor zetakine is
an extracellular domain containing a mutein of IL-13;
spacer;
a transmembrane domain; and an intracellular signaling region,
said spacer consists of IgG4 hinge- CH3 consisting of 119 amino acids in length and comprising an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10;
the spacer links the mutein to the transmembrane domain, and the transmembrane domain links the spacer to the intracellular signaling region;
nucleic acid. 2. The nucleic acid of claim 1, wherein said mutein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:17. 3. The nucleic acid of claim 1 or 2, wherein said mutein comprises the amino acid sequence shown in SEQ ID NO:17. The nucleic acid of any one of claims 1-3 , wherein said transmembrane domain comprises a CD28 transmembrane domain. 5. The nucleic acid of any one of claims 1-4 , wherein the intracellular signaling region comprises a co-stimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, NKG2C, B7-H3, and combinations thereof, in combination with all or part of the CD3ζ domain. The nucleic acid of any one of claims 1-5 , wherein said intracellular signaling region comprises a portion of the CD3ζ domain and a co-stimulatory portion of the 4-1BB domain. The nucleic acid of any one of claims 1-6 , further comprising a sequence encoding a marker protein. 8. The nucleic acid of claim 7 , wherein said marker protein comprises truncated epidermal growth factor receptor or dihydrofolate reductase (DHFR). 9. The nucleic acid of claim 8 , wherein said DHFR is a double mutant of DHFR (DHFRdm). 10. The nucleic acid of claim 9 , wherein said DHFRdm comprises an L22F amino acid mutation and an F31S amino acid mutation. The nucleic acid of any one of claims 1-10 , further comprising a ribosome skipping sequence. 12. The nucleic acid of claim 11 , wherein said ribosome skip sequence comprises P2A or T2A. An expression vector comprising a nucleic acid according to any one of claims 1-12 . 14. The expression vector of claim 13 , which is a viral vector. 15. The expression vector according to claim 13 or 14 , which is a lentiviral vector or an adenoviral vector. A zetakine that is a receptor targeted by an IL-13 mutein encoded by the nucleic acid of any one of claims 1-12 . A cell containing the nucleic acid according to any one of claims 1-12 . 18. The cell of claim 17 , which is a lymphocyte. 19. The cell of claim 18 , wherein said lymphocyte has a CD45RA-, CD45RO+ and CD62L+ phenotype. A cell according to any one of claims 17 to 19 , which is a naive CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell or a CD45RO+CD62L+CD8+ T cell. A cell according to any one of claims 17 to 19 , which is a naive CD4+ T cell, a central memory CD4+ T cell, an effector memory CD4+ T cell or a CD45RA+CD62L+CD4+CD45RO- T cell. 18. The cell of claim 17 , which is a progenitor T cell. 23. The cells of claim 22 , wherein said progenitor T cells are hematopoietic stem cells. A composition comprising the cells of any one of claims 17-23 and a pharmaceutically acceptable carrier. A method of making a genetically engineered cell containing the nucleic acid of any one of claims 1-12 , comprising:
introducing a nucleic acid according to any one of claims 1 to 12 into a cell to produce a genetically engineered cell;
culturing said genetically engineered cells in the presence of anti-CD3 and/or anti-CD28 antibodies and at least one homeostatic cytokine; and enriching said genetically engineered cells after culturing. 26. The method of claim 25 , wherein said nucleic acid further comprises a sequence encoding a marker protein, and said enriching step comprises culturing said genetically engineered cells after culturing with a reagent that selects for marker protein expression. 27. The method of claim 25 or 26 , wherein said cells are lymphocytes. 28. The method of claim 27 , wherein said lymphocytes have a CD45RA-, CD45RO+ and CD62L+ phenotype. 29. The method of any one of claims 25-28 , wherein said cells are naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells or CD45RO+CD62L+CD8+ T cells. 29. The method of any one of claims 25-28 , wherein said cells are naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells or CD45RA+CD62L+CD4+CD45RO- T cells. 27. The method of claim 25 or 26 , wherein said cells are precursor T cells. 32. The method of claim 31 , wherein said progenitor T cells are hematopoietic stem cells. 33. The method of any one of claims 25-32 , wherein said at least one homeostatic cytokine is IL-15, IL-7, IL- 21 or a combination thereof. A method according to any one of claims 26 to 33 , wherein said marker protein is truncated epidermal growth factor receptor. Use of the cells according to any one of claims 17 to 23 in the manufacture of medicaments for treating or suppressing cancer expressing IL-13 receptor α2 (IL-13Ra2). 36. Use according to claim 35 , wherein said cancer comprises brain cancer. 37. Use according to claim 36 , wherein said brain cancer is glioma, glioblastoma or glioblastoma multiforme. Use according to any one of claims 35 to 37 , wherein said cancer is an IL-13Rα-positive malignant tumor. 25. The composition according to claim 24 , which is for treating cancer. 40. The composition of claim 39 , wherein said cancer is an IL13Rα-positive malignant tumor. 41. The composition of claim 39 or 40 , wherein said cancer comprises brain cancer. 42. The composition of claim 41 , wherein said brain cancer is glioma, glioblastoma or glioblastoma multiforme. 43. The composition of any one of claims 39-42 for administration to a subject undergoing therapy selected from chemotherapy and radiotherapy. 44. The composition of claim 43 , wherein said chemotherapy comprises electrochemotherapy, alkylating agents, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators, checkpoint inhibitors, or combinations thereof. 44. The composition of claim 43 , wherein said chemotherapy comprises 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, or combinations thereof. 46. The composition according to any one of claims 43-45 , which is for administration to a mammal. 47. The composition according to any one of claims 43-46 , which is for administration to humans.

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